Many clinical applications call for stratification of patients by molecular (and/or other) attributes. For example, to develop or administer personalized therapies, patients may be selected for clinical trials or drug development programs in accordance with molecular attribute profiles that provide a differential diagnosis, such as for many cancers, or indicate the efficacy and/or safety of therapy (see e.g. Doehner2010, Kurose2012).
Further, many patients with cytopenias require regular transfusion. For example, patients with anemia caused by renal disease, or by hematologic disorders including leukemia, sickle cell anemia, or thalassemia, require regular red blood cell transfusions, and especially the care for the chronically transfusion-dependent generates substantial cost (Wayne2000). Likewise, patients with certain hematologic disorders including acute leukemias and certain cases of myelodysplatic syndrome who develop thrombocytopenia require extensive platelet transfusion support, once again at substantial expense (Meehan2000). Periodic transfusion often leads to progressive alloimmunization against an increasing number of antigenic determinants displayed on the donor cells, be they red cells (Castro2002) or platelets (TRAP1997).
Platelets: Human Leukocyte Antigens (“HLA”) Class I, Human Platelet Antigens (“HPA”)—Patients receiving therapy for hematologic malignancies consume more than 40% of the approximately 2.1 million single donor units (or equivalents) collected in the US as of 2013 (AABB2013). Many patients have antibodies, formed in response to prior allogeneic exposure during pregnancy or previous transfusion, and others develop antibodies during treatment, and these antibodies mediate the accelerated clearance of transfused cells, leading to a poor response to transfusion and excess platelet consumption as well as excess utilization of clinical services, and extended in-hospital stays, especially for patients who respond to transfusion (Meehan2000).
In part, this state of affairs reflects the logistical difficulty of identifying suitable platelet donors quickly in view of the short expiration dating for platelets. Random searches that identify prospective donors by a negative serological cross-match, are time-consuming and, at best, will exclude as unsuitable only those prospective donors with cognate epitopes to existing antibodies, but will not identify allo-epitopes that may lead to the formation of new antibodies. The genotyping of HLA, though long since a standard approach to matching stem cell recipients and donors, as currently practiced, is complex and slow, and the prevailing strategy of procuring stem cells has been to maintain large registries of volunteers who are genotyped at registration, an expensive propositions the vast majority of these volunteers will never called. Creating large registries of potential platelet donors clearly is impractical for the routine procurement of suitable platelets, given the large demand, and the time constraints imposed by the platelet expiration dating.
Red Blood Cells: Human Erythrocyte Antigens (“HEA”)—For sickle cell patients, stroke is a major risk factor, and timely (hence chronic) transfusion has been shown to be very effective in reducing that risk (eg. Lee2006). The commercial introduction of routine genotyping into donor centers and hospital transfusion services, a decade ago (Hashmi2005, Hashmi2007, Moulds2011), has greatly facilitated the procurement of suitable red cells especially for transfusion-dependent patients with multiple antibodies, a common side effect of chronic transfusion of sickle cell anemia and thalassemia patients (Castro2002, Pham2011, Chou2013). However, notwithstanding its commercial availability in several formats, genotyping, given its perceived high cost and complexity—which may require special training and in some cases certification—has been limited, in practice, to special situations that are not readily handled by serology. Serology, largely automated, has otherwise remained the “work horse” in the pre-transfusion setting, especially for large-scale “pre-selection” of candidate donors.
Finding candidate cells with desirable molecular attributes, usually in the form of a set of cell surface markers (expressed or not expressed), or a set of antigenic determinants associated with antigens such as HLA, is a search problem. The prevalent format of genotyping represents a “brute force” solution that is ill-suited to scale up. Thus, to identify, in accordance with this format, donors who do not express the RBC antigens E, V and Fya, say, one first genotypes all candidate donors at hand—one at a time—for an entire set of alleles (as in, say, BioArray Solutions' “HEA PreciseType” test, see website at Immucor, Inc.), then looks for instances, if any, that lack the specified antigens. As this attribute pattern—“E- & V- & Fya-”—is not a common one, many of the genotype determinations will be of no value, and unless they address other instances of pending requests, the investment made in those determinations may be lost.
Consequently, to reduce genotyping expenditures, many hospital transfusion services supporting transfusion-dependent patients have resorted to extraordinary measures such as pairing individual patients with special (“buddy”) donors on whose continuing kindness they count for a vital part of their patient care; all the while generating tens of thousands of dollars in annual expenses for other aspects of care for the very same patients, particularly laboratory charges and “spend” for iron chelators (Wayne2000).
In the hematology/oncology setting, the situation is worse. Unless patients become non-responsive to platelet transfusion, the procurement and selection of platelets, in order to ensure hemostasis and to maintain vascular integrity, remains largely uninformed by concerns about the risk of allogeneic exposure to antigens displayed on platelets, notably HLA (class I) and HPA, and its clinical and financial consequences
On the supply side, many practitioners rely on serological methods, preferably in an automated format, to “pre-screen” candidate units for genotyping, usually representing a fraction of no more than a few percent. However, for all but the most common red cell antigens such as C, E and K, this approach must rely on a limited (and expensive) supply of reagents. In addition, it has the disadvantage that it not only proceeds one sample at a time, but also one antigen at a time, and therefore requires elaborate sample handling and tracking. In the alternative, many practitioners, in lieu of extensive pre-screening, invoke simple heuristics for pre-selection, for example, on the basis of major blood type and/or declared ethnic background. Many also favor repeat donors, thereby in some cases severely narrowing the distribution of available antigen profiles.
To overcome the limitations of current approaches to large-scale genotyping generally, and to the routine procurement of blood cells or other cells with specific antigen and genotype profiles, a process is needed that: (i) enables the effective scale-up of genotyping to survey and profile large numbers of samples, and (ii) does so in a manner ensuring superior performance over the prevailing “brute-force” search strategy, preferably while decreasing, and certainly without unduly increasing, the cost per “hit”. An effective search process, related to “Nucleic Acid Sieving” (U.S. Pat. No. 8,932,989 and US Publ'n No. 2015/0315568), “Allele Profiling” (US Publ'n Nos. 20130029857 and 2015/0376693) and “Attribute Profiling” (U.S. Pat. No. 9,133,567), all of which are incorporated by reference, is disclosed herein.